Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µm, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca++. We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

A survey of Pisum genotypes for seed trypsin inhibitors revealed a tenfold range in the extent of inhibition. Approximately 90% of trypsin inhibitory activity was associated with two albumin fractions in selected variant lines. The differences among extreme variants were consistent in three environments, between two sources of trypsin tested and whether expressed on a unit protein or dry weight basis.

A study of the appearance of trypsin inhibitors during seed development in selected highand low-inhibitor lines showed differences in the accumulation pattern of active inhibitors. An endogenous protease was identified in Pisum seed protein preparations, whose in vitro trypsin-like activity was predominant in protein from early stages of seed development, when little or no trypsin inhibitor was present. However, there was no correlation between the amount of this protease and the extent of trypsin inhibitory activity in lines that varied for inhibitor content.

There is a carboxypeptidase in mung bean seeds that is localized in the protein bodies, the same vacuoles in which seed storage proteins are sequestered. This carboxypeptidase, called carboxypeptidase I (or Cpase I) has been purified by a series of ion-exchange and gel filtration columns. The pure enzyme consists of a single polypeptide chain with a MW of 41700 by SDS-PAGE or 42000 by size-exclusion HPLC. It has a pl of 4.36 and is a serine carboxypeptidase as shown by its inactivation by phenylmethylsulfonyl fluoride, and its resistance to other proteolytic inhibitory reagents. A survey of its activity with Cbz-dipeptides shows preference for C-terminal amino acids that are large, hydrophobic residues, and a small aliphatic amino acid such as alanine, but not glycine, at the penultimate amino acid residue. Cpase I can convert a trypsin inhibitor of the mung bean to its proteolytic intermediate lacking four amino acid residues at its C-terminus. This proteolytic intermediate is detected in the mung bean cotyledons during early growth. Levels of both the activity and immunological cross-reacting forms of this enzyme start high and decrease during early growth.

The effect of feeding rats purified cowpea (Vigna unguiculata Walp.) trypsin (EC 3.4.21.4) inhibitor in a semi-synthetic high-quality diet based on lactalbumin (10 g inhibitor/kg) for 10 d was a moderate reduction in the weight gain of rats in comparison with controls, despite an identical food intake in the two groups. The reduction in the growth rate was about 20% on a live weight basis. However, the corresponding value calculated from the weight of dry carcasses was less, only about 7%, probably because the water content of the body of the two groups of rats was different. Although most of the cowpea trypsin inhibitor (CpTI) was rapidly broken down in the digestive tract, its inclusion in the diet led to a slight, though significant, increase in the nitrogen content of faeces but not of urine. Accordingly, the net protein utilization of rats fed on inhibitor-containing diets was also slightly depressed while their energy expenditure was elevated. In agreement with results obtained for the protease inhibitors of soya bean, the slight anti-nutritional effects of CpTI were probably due mainly to the stimulation of the growth and metabolism of the pancreas. Thus, the nutritional penalty for increased insect-resistance after the transfer of the cowpea trypsin inhibitor gene into food plants is slight in the short-term.